1. Field of the Invention
This invention relates to a stable, homogeneous, biologically active human relaxin formulation and to a method of treating mammals with such formulation.
2. Description of the Background Art
Mature human relaxin is an ovarian hormonal peptide of approximately 6000 daltons known to be responsible for remodelling the reproductive tract before parturition, thus facilitating the birth process. Hisaw, F. L., Pros. Soc. Exp. Biol. Med., 23, 661-663 (1926); Schwabe, C. et al., Biochem. Biophys. Res. Comm., 75: 503-570 (1977); James, R. et al., Nature, 262: 544-546 (1977). This protein appears to modulate the restructuring of connective tissues in target organs to obtain the required changes in organ structure during pregnancy and parturition. Some of the important roles for relaxin as a pregnancy hormone include inhibition of premature labor and cervical ripening at parturition.
While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, G. D., Endocrine Reviews, 3: 62-90 (1982) and Weiss, G., Ann. Rev. Physiol., 46:43-52 (1984).
Relaxin has been purified from a variety of species including porcine, murine, equine, shark, tiger, rat, dogfish, and human, and shows at least primary and secondary structural homology to insulin and the insulin-like growth factor. In the human, relaxin is found in most abundance in the corpora lutea (CL) of pregnancy.
Two human gene forms have been identified by genomic cloning using probes from the porcine relaxin gene (Hudson, P. et al., Nature, 301: 628-631 [1984] and Hudson, P. et al., The EMBO Journal, 3: 2333-2339 [1984]), although only one of these gene forms (H2) has been found to be transcribed in CL. It is unclear whether the other gene is expressed at another tissue site, or whether it represents a pseudo-gene. The two human relaxin genes show considerable nucleotide and amino acid homology to each other, particularly in the B and C peptides. However, there are some notable regions of sequence divergence, particularly in the amino-terminal region of both A- and B-chains.
Similar to all species examined, the primary translation product of H2 relaxin is a preprorelaxin consisting of a 24 amino acid signal sequence followed by a B chain of about 29 amino acids, a connecting peptide of 104-107 amino acids, and an A chain of about 24 amino acids.
The fact that H2 relaxin is synthesized and expressed in the ovary suggests that this is the sequence that is involved in the physiology of pregnancy. When synthetic human relaxin (H2) and certain human relaxin analogs were tested for biological activity, the tests revealed a relaxin core necessary for biological activity as well as certain amino acid substitutions for methionine that did not affect biological activity. Johnston et al. , in Peptides: Structure and Function, Proc. Ninth American Peptide Symposium, Deber, C. M. et al. (eds.) (Pierce Chem. Co. 1985).
Various methods for applying or introducing porcine relaxin as a drug have been published, including intravenous and intramuscular administrations. Birnberg and Abitol, Obstet. & Gynecol., 10: 366-370 (1957); Eichner et al., Am. J. Obstet. Gyn., 71: 1035-1048 (1956). Additionally, porcine relaxin has been used topically in human clinical trials for ripening of the cervix and induction of labor. MacLennan et al., Obstetrics & Gynecology, 68: 598-601 (1986); MacLennan et al., The Lancet, i:220-223 (1980); MacLennan et al., Obstetrics & Gynecology, 58: 601-604 (1981); MacLennan et al., Aust. NZ J. Obstet Gynaec., 25: 68-71 (1985). In these studies the porcine relaxin in distilled water was mixed with water-soluble methylcellulose granules to make a viscous gel. A 2-mg dose was found to improve the cervical score. MacLennan et al. (1981), supra.
In addition, porcine relaxin has been molded into pellets made from polyethylene glycol and used to ripen the human cervix. Evans et al., Am. J. Obstet. Gynecol.,147: 410-414 (1983). Porcine relaxin also has been formulated by suspension in a sterile K-Y gel and phosphate-buffered saline and infused into the cervical os by a sterile insemination pipette. Dilatation of the cervix increased within 8 hours after infusion of 3000 units of relaxin in the cervical os. Perezgrovas and Anderson, Biology of Reproduction, 26:765-776 (1982).
European Pat. Publ. No. 86,649 published Aug. 24, 1983 discloses how to prepare porcine preprorelaxin, porcine prorelaxin, and porcine relaxin. Australian Pat. No. 561,670 issued Aug. 26, 1987, European Pat. Publ. No. 68,375 published Jan. 5, 1983, and Haley et al. , DNA, 1:155-162 (1982) disclose how to prepare porcine relaxin.
European Pat. Publ. No. 101,309 published Feb. 22, 1984 and U.S. Pat. No. 4,758,516 issued Jul. 19, 1988 respectively disclose the molecular cloning and characterization of a gene sequence coding for human HI-relaxin and human H2-relaxin and analogs thereof. European Pat. Publ. No. 260,149 published Mar. 16, 1988 discloses formulations of human prorelaxin. U.S. Pat. No. 4,267,101 issued May 12, 1981 discloses a process for obtaining human relaxin from fetal membranes. European Pat. Publ. No. 251,615 published Jan. 7, 1988 discloses how to combine reduced human relaxin A-chain or analog and reduced human relaxin B-chain or analog under conditions that include a pH greater than about 7.0.
A process for obtaining human relaxin from fetal membranes is disclosed in U.S. Pat. No. 4,267,101. Sherwood, in The Physiology of Reproduction, ed. by Knobil and Neill, (New York, Raven Press,1988), p. 585-673, discloses at p. 587 that almost all of the procedures for extraction and isolation of relaxin take advantage of the stability of relaxin in acidic solvents. In addition, on page 588 it is noted that strongly acidic medium for extraction of relaxin minimizes proteolysis both by the low pH per se and by precipitation of high-molecular-weight proteases. Eldridge and Fields, Endocrinology, 117: 2512-2519 (1985) reported that rabbit relaxin has an isoelectric point, as determined by isoelectrofocusing, of about 6.5. This is in contrast to the findings of Fields et al, Ann. NY Acad. Sci, 380: 75 (1982), who discovered a much higher isoelectric point for rabbit relaxin.
In addition, copending U.S. application Ser. No. 07/84,255 filed Aug. 10, 1987 discloses in Example 4 dialyzation of refolded relaxin samples into phosphate buffer, Tween 20, or 0.1% acetic acid.
Moreover, U.S. Pat. No. 2,964,448, issued Dec. 13, 1960, discloses injectable relaxin compositions comprising relaxin in an injectable oil, preferably of low acidity, together with a fatty acid salt of aluminum.
It is an object of the present invention to provide a biologically active, stable, homogeneous formulation of human relaxin for use in therapeutic, i.e., topical or parenteral, applications. Another object of this invention is to provide a formulation permitting storage for a long period of time in a liquid state, thereby facilitating storage and shipping prior to administration. Still another object is to reduce aggregation and degradation of human relaxin and to provide a formulation thereof that is resistant to degradation resulting from fluctuations in temperature. Yet another object is the formulation of human relaxin as a gel for cervical applications. A further object is to provide a formulation of human relaxin that has additional therapeutic and physical/chemical advantages.
These and other objects of this invention will be apparent from consideration of this specification as a whole.